1. Field of the Invention
The invention relates to a process for producing orchid seedlings, in particular the seedlings of moth orchids (genus Phalaenopsis), by static liquid culture, in which orchid seeds are suspended in a liquid medium suitable for the germination and growth thereof, and the depth of the thus obtained seed suspension in a culture container is set to be within a predetermined range at the onset of culture.
2. Description of the Related Art
The major export crops of Taiwan are orchids, in particular moth orchids (genus Phalaenopsis). Orchids are not only the number one export commodity of Taiwan, they are also very popular in Taiwan's domestic market. In Taiwan, over 90 million seedlings of Phalaenopsis are produced each year, of which 60% are seedlings developed from seeds. The business of breeding orchid seedlings is therefore a very lucrative one. However, in order to maintain dominance in the export market, the techniques of tissue culturing orchid seedlings have to be improved so as to lower costs and upgrade quality.
At present, solidified agar media are used in the production of orchid flask seedlings. Agar accounts for about 30–40% of the cost of the culture medium materials, and is the most expensive ingredient of the culture medium formulas. Besides, the step of melting agar is the most time-consuming procedure in the preparation of the medium. In addition, in the cultivation of orchid seedlings, sowing of seedlings in vitro is still the most widely used method of production. However, the seedling production processes are largely traditional. For instance, glass flasks with rubber stoppers, which do not provide good ventilation, are used as culture containers. In addition, high density sowing and high density subculture are adopted in the culture of seedlings. Non-uniform growth of seedlings is a common phenomenon on a production line that adopts the traditional seedling production process, and multi-stage screening is therefore required. During the process of subculturing, oftentimes, many smaller seedlings have to be discarded, resulting in a waste of seedlings and higher labor costs. Therefore, improving the seedling production process is an important factor to be taken into consideration in the attempt to reduce orchid seedling production costs.
Furthermore, it is reported that agar, when used as a gelling agent to prepare a solid culture medium, will affect the osmotic potential of the culture medium, thereby affecting the absorption of nutrients by an explant (von Arnold and T Eriksson (1984), Plant Cell, Tissue and Organ Culture, 3:257–264). In other studies, it has been pointed out that the amount of cytokinin absorbed by an explant is in an inverse correlation with the rigidity of the gel of the culture medium (Debergh, P. C. and L. J. Maene (1981), Sci. Hort., 14:335–345; Bomman, C. H. and T. C. Vogelmann (1984), Physiol. Plant., 61:501–512), thereby affecting the multiplication rate.
Liquid culture has been proven to be capable of achieving an explant multiplication rate higher than that achievable by solid culture for many plants, such as roses (Chu, C. Y., M. Y. Wang (1995), “Effect of culture medium on growth of tissue-cultured explants,” Journal of Agriculture and Forestry, 44 (4):71–77), rhododendrons (Douglas, G. C. (1984), Sci. Hort., 24: 337–347), pears (Viseur, J. (1987), Acta Hort., 212: 117–124), and conifers (Pâques, M. J. et al. (1992), Acta Hort., 319: 95–100), etc.
However, plant seedlings are susceptible to hyperhydricity (or vitrification) after being grown in a liquid medium for a long time (Etienne, H. and M. Berthouly (2002), Plant Cell, Tissue and Organ Culture, 69: 215–231). Many studies showed that once an explant was observed to develop hyperhydricity, it was difficult to recover the explant to its normal state. Although increasing the rigidity of the culture medium is an effective way to restore the hyperhydric explant to its normal state, it is not suitable for certain plants, such as protocorm-like-bodies (PLBs) of Doritaenopsis (Zhou, T. S. (1995), Plant Cell Reports, 15:181–185).
The use of a liquid medium in conjunction with shaking, rotation or direct aeration of the culture medium to propagate plants was proposed in the cultivation of Begonia×hiemalis in the early years (Takayama, S. et al. (1981), Plant and Cell Physiol., 22:461–467). In the recent decades, there have been reports on the successful propagation of other plants, such as lilies (Takayama, S. et al. (1991), Automated propagation of microbulbs of lilies. In: Wasil I K (Ed.) Cell Culture and Somatic Cell Genetics of Plants. Vol. 8:111–131. Academic Press, Inc.), Nerine (Ziv, M. et al. (1994), Plant Cell, Tissue and Organ Culture, 39:109–115; Lilien-Kipnis, H. et al. (1994), Plant Cell, Tissue and Org. Cult., 39:117–123), carrots (Onishi, N. et al. (1994), Plant Cell, Tissue and Org. Cult., 39:137–145; Osuga, K. et al. (1994), Plant Cell, Tissue and Org. Cult., 39:125–135) and Euphorbia pulcherrima (Luttman, R. et al. (1994), Plant Cell, Tissue and Org. Cult., 39:157–170).
As for orchids, there are reports pointing out that the use of liquid culture can increase the rate of proliferation of protocorms (Adelberg, J. W. et al., (1992), Amer. Orch. Soc. Bull., 61:688–695; Adelberg. J. W. et al. (1997), Plant Cell, Tissue Org. Cult., 48:1–7); Lakshmanan, P. et al. (1995), Plant Cell Rep., 14:510–514; Young, P. S. et al. (2000), Plant Cell, Tissue and Org. Cult., 63:67–72). However, proliferation of protocorms from seedlings has to be avoided so as not to delay differentiation of leaves and so as not to result in mutation of the variety, which may affect assessment of the offspring trait distribution. At present, the applicants are not aware of any study or report on the production of orchid seedlings by sowing in a static liquid suspension.
Occurrence of vitrification in explants is mainly caused by an increase in the water potential. That is, an increase in the water usable by the explant leads to fast abnormal growth so that the explant lacks lignin and cuticle, thereby resulting in a glassy appearance, and the content of chlorophyll drops, thereby resulting in production of ethylene (Phan, C. T. and P. Hegedus (1986), Plant Cell Tissue and Org. Cult., 6:83–94). High cytokinin concentration and low agar concentration will also promote generation of vitrificated explants. Although addition of phloroglucinol reagent to the culture medium (Phan, C. T. and P. Hegedus (1986), supra), or use of cold treatment (Boxus, P. (1978), Rapport d'Activite Compter Rendus, Gembloux, Belgique. pp. 126–127) is capable of restoring the vitrified explant to normal growth, the best way is to lower the concentration of cytokinin and increase the amount of agar or to increase air circulation in the vessel so as to prevent production of vitrified explants (Pasqualetto, M. et al. (1986), Acta Hort., 319: 95–100). In the production of flask seedlings of Phalaenopsis, since there is no need for proliferation of protocorms, cytokinin is rarely used. Therefore, if a liquid medium is to be used to culture flask seedlings, the water potential and the air ventilation of the culture container must be considered.
At present, solid culture media are widely used for the production of flask seedlings of Phalaenopsis, and glass flasks are used as culture containers, which are sealed by a rubber stopper with a hole plugged by cotton wool. Take the production of Phalaenopsis seedlings as an example. Flask transfer of the seedlings has to be conducted for at least two times after seed sowing, and it would take around one year before the seedlings can be removed from the flasks for planting. Since cultivation of flask seedlings of Phalaenopsis requires three to four times of flask transfer operations, which account for about 65–70% of the labor cost, if the flask transfer operations in the seedling production can be simplified, or the labor costs needed for conducting one to two times of flask transfer can even be dispensed with, the production costs can be reduced considerably. Furthermore, if a liquid medium is used to culture orchid flask seedlings, since solidification of the culture medium is not required, the preparation cost can be lowered. Moreover, the use of a liquid medium to produce orchid seedlings can result in uniform growth of the flask seedlings, which facilitates the grading of seedlings in the subsequent subculturing process, and simplifies and speeds up the operating procedures of subculturing, thereby reducing labor costs and upgrading the quality of flask seedlings. Therefore, if the problem of vitrification associated with the use of liquid media can be overcome, the competitive edge of orchid flask seedling production can be enhanced to a large extent.